The ability to eliminate potentially lethal or carcinogenic damage from the genetic pool is a crucial step in an organism's response to injury by radiation or chemicals. The objective of the proposed research is to identify and characterize the enzymes and reactions involved in excision repair of DNA in human cells exposed to either radiation or alkylating agents. My efforts will continue to focus on the critical first step in the excision repair pathway, namely recognition of and strand incision at specific lesions in DNA. During the research period just completed, an endonuclease was partially purified from human lymphoblasts (CCRF-CEM line) and was shown to act specifically on lesions in DNA treated with either UV, x-rays or methyl methanesulfonate (MMS). This UV-X-MMS enzyme will be characterized further, with emphasis on establishing that it is in fact an endonuclease and not a N-glycosidase. Purification of the enzyme will be continued to (1) resolve separate activities against DNA treated with either UV, x-rays or MMS and (2) to separate a DNA-binding activity presently associated with the enzyme. Should the binding activity be due to a discrete protein, its specificity for DNA lesions induced by different agents, and its interaction with UV-X-MMS enzyme will be determined. The functional significance of th UV-X-MMS enzyme in vivo will be sought by quantitating enzyme-susceptible lesions in DNA from x-irradiated skin fibroblasts obtained from both normal individuals and those with ataxia telangiectasia (AT), an inherited condition characterized in part by an extreme sensitivity to x-radiation. Enzyme-induced strand breaks will be measured by velocity sedimentation analysis in alkaline sucrose gradients. Disappearance of these sites during post-irradiation incubation of the normal cells will indicate excision of damaged bases; x-ray-sensitive AT cells are likely deficient in this function. An absence or a defect in the UV-X-MMS enzyme in AT cells will be further proof that this enzyme has an important function in repair of DNA in man. Knowledge gained from this proposal will lead to a clearer understanding of the mechanisms and importance of excision repair of specific lesions in human DNA, especially as related to genetic disorders characterized by sensitivity to sunlight and an increased frequency of cancer. F (Text Truncated - Exceeds Capacity)